CN112553268B - Method and device for synthesizing trehalose by ultrasound-assisted enzyme - Google Patents

Abstract

The invention relates to the field of enzyme preparation, and specifically discloses a method and device for synthesizing trehalose with ultrasound-assisted enzyme, which includes the following steps: adding water, starch, and α-amylase to a reaction kettle for mixing, then adjusting the pH value, and then adding Calcium chloride water, start the stirring shaft of the reactor to stir the above mixture, multiple ultrasonic transducers, heaters and thermometers, start the circulation path to transfer the mixture at the bottom of the reactor outward, crush and filter, and then return it to the reactor. within; heat the mixture in stages and keep it warm; stop heating when the liquefied DE value reaches 15, cool the mixture to normal temperature while stirring and circulating, adjust the pH value of the mixture, and then add pullulanase, MTSase, MTHase and α/β-CGTase , reheat and keep at 45°C, adjust the pH value of the mixture, and finally add glucoamylase for mixing to finally obtain the product. Ultrasound-assisted enzymatic synthesis of trehalose has a significant effect on improving the conversion rate of trehalose synthesized from rice starch as the basic raw material.

Description

A method and device for ultrasonic-assisted enzymatic synthesis of trehalose

Technical field

The invention belongs to the field of enzyme preparation, and in particular relates to a method and device for synthesizing trehalose with an ultrasound-assisted enzyme.

Background technique

Some insects and plants that have lived in the desert for many years are almost dried out and dehydrated in the high temperature at noon, and are in a state of physiological suspended animation. However, once rainfall replenishes water, they can be revived within a few hours. Research on these cryptobionts by scholars at the University of Cambridge in the UK shows that this resurrection phenomenon is due to the presence of high concentrations of trehalose in their bodies. In addition, some frogs and other organisms can survive in severe cold conditions. The important reason is also the protective effect of trehalose on cells. The stress tolerance displayed by many organisms under stressful environments (such as starvation, high temperature, freezing, drying, hypertonicity, radiation, toxic substances, etc.) is directly related to the trehalose content in the body. Yeast can still be resurrected after removing 90% of its water and rehydrating it. Under long-term starvation conditions, the survival rate of yeast depends on the trehalose content in the body. It has been reported that heat shock and ethanol shock treatment of yeast can cause a significant increase in intracellular trehalose content.

Trehalose is a substance that is highly resistant to stress caused by environmental changes and is a typical stress metabolite in organisms. In various harsh environments, trehalose has shown good protective effects on species' biofilms, proteins, nucleic acids and other biological macromolecules. The latest research shows that exogenous trehalose has a good non-specific protective effect, so some people call trehalose the "sugar of life."

With the improvement of social living standards, the demand for trehalose is constantly increasing. Due to the low yield of trehalose and the complicated production process, the product price is still high, which limits the promotion and application of trehalose. How to increase the yield of trehalose, how to improve the production method, and how to reduce the production cost are important topics in the research of trehalose.

Contents of the invention

The object of the present invention is to provide a method and device for ultrasound-assisted enzyme synthesis of trehalose to improve the efficiency of synthesizing trehalose.

In order to achieve the above object, the present invention provides a method for ultrasound-assisted enzyme synthesis of trehalose, which includes the following steps:

S1. Add water, 150g/L rice starch, and α-amylase with an activity of 15U/g into the reaction kettle, adjust the pH value to 6.2 with acid, and add anhydrous calcium chloride at the same time to make the final concentration 2g/L. ;

S2. Start the stirring shaft of the reactor to stir the above mixture. At the same time, start the ultrasonic transducer in the reactor that is submerged in the above mixture. The actual power of the ultrasonic transducer is 100-200w and the vibration frequency is 30kHz. Start the ultrasonic transducer in the reactor. The heater starts the circulation path to transfer the mixture at the bottom of the reactor outward, crush, filter, and then return it to the reactor;

S3. Heat the mixture to 75°C. When the temperature of the mixture equals 75°C, keep it warm for a certain period of time. Heat the mixture to 90°C and keep it warm for a certain period of time;

S4. When the liquefaction DE value reaches 15, stop heating, stir and circulate the mixture to cool to normal temperature, and adjust the pH of the mixture to 6;

S5. Add 0.75U/ml pullulanase, add 2.4U/ml MTSase and MTHase, add α/β-CGTase with a concentration of 1.4U/ml, reheat and incubate at 45°C for 25-30h, adjust The pH of the mixture is 4.5;

S6. Add glucoamylase, raise the temperature to 60°C and keep it incubated for 10-12 hours;

S7. Obtain the product.

As an improvement to the above scheme, the MTSase is fermented by B.subtilis/pHY300PLK-PgsiB-Y, the MTHase is fermented by B.subtilis/pHY300PLK-Pxyl-Z, and the α/β-CGTase is fermented by B .subtilis/pHY300PLK- Fermented from PhpaII-PamyQ'-CGT.

As an improvement to the above solution, in step S4, the ventilation port of the reaction kettle is opened to dissipate heat, and the mixture is naturally cooled during stirring and circulation.

In order to achieve the above object, the present invention provides a device for ultrasonic-assisted enzymatic synthesis of trehalose, which includes a reaction kettle. The inside of the reaction kettle is provided with a stirring paddle, a heater and a thermometer, and the upper end of the reaction kettle is provided with a liquid inlet. The lower end of the reaction kettle has a drain port and a valve thereof. The lower end of the reaction kettle has a drain port and a valve thereof. The characteristic is that the drain port at the lower end of the reactor is provided with two branches, namely the first branch and the second branch. The first branch is used to discharge the mixture to the outside world, the second branch returns to the liquid inlet of the reaction kettle, the second branch is provided with a pump body and a pulverizer, and the side wall of the reaction kettle is provided with There are several ultrasonic transducers, and the transmitting end of each ultrasonic transducer faces the inside of the reactor.

As an improvement to the above solution, the second branch is provided with a third branch and its valve at a position away from the pulverizer, for passing the mixture that does not require further pulverization.

As an improvement to the above solution, a sampling port is provided on the second branch road.

As an improvement to the above solution, the blades of the stirring paddle are spiral blades. When the stirring paddle rotates, the spiral blades drive the mixture to rise, and the mixture descends from a position near the inside of the reaction kettle.

The invention has the following beneficial effects:

1. The ultrasonic-assisted enzymatic synthesis of trehalose effectively improves the trehalose conversion rate, saves resources, reduces costs, and has high trehalose yield, which is conducive to industrial production and application.

2. Starch liquefaction and enzyme-catalyzed synthesis reactions are carried out in the same reactor, eliminating the trouble of material transfer, saving labor time and improving efficiency.

3. Under the conditions of ultra-fine grinding and material circulation, the starch is liquefied, the liquefaction efficiency is improved, heating and cooling are uniform, the slurry can circulate up and down, and there is no mixing dead zone.

Description of the drawings

Figure 1 is a structural diagram of a reactor and its interior under an embodiment.

Explanation of reference signs: 10. Reactor; 11. Liquid inlet; 21. Liquid outlet; 13. Stirring paddle; 14. Heater; 15. Thermometer; 16. Ultrasonic transducer; 21. Pump body; 22. Crusher; 31. First branch; 32. Second branch; 33. Third branch; 34. Sampling port.

Detailed ways

The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

Referring to Figure 1, the present invention discloses a method and device for ultrasound-assisted enzymatic synthesis of trehalose.

The device is based on the existing reaction kettle 10. The inside of the reaction kettle 10 has a stirring paddle 13, a heater 14 and a thermometer 15. The upper end of the reaction kettle 10 has a liquid inlet 11 and its valve. The lower end of the reaction kettle 10 has a drain port 12 and its valve. Furthermore, the lower end drain port 12 of the reaction kettle 10 is provided with two branches, namely a first branch 31 and a second branch 32. The first branch 31 is used to discharge the mixture to the outside, and the third branch is used to discharge the mixture to the outside. The second branch 32 returns to the liquid inlet 11 of the reaction kettle 10. The second branch 32 is provided with a pump body 21 and a pulverizer 22. The side wall of the reaction kettle 10 is provided with a number of ultrasonic transducers 16. The transmitting end of each ultrasonic transducer 16 faces the inside of the reactor 10 . The arrows in Figure 1 indicate the flow direction of the mixture. After the mixture is stirred in the reaction kettle 10, it is driven by the pump 21 and returns to the liquid inlet 11 along the second branch 32 on the left, and then is put back into the reaction kettle 10.

In this embodiment, several holes are drilled in the side wall of the reaction kettle 10 for inserting several ultrasonic transducers 16, and then the inside of the reaction kettle 10 is sealed, and a sealing ring is provided at the gap. Each ultrasonic transducer 16 is evenly distributed to provide uniform vibration, and the wires and signal lines of each ultrasonic transducer 16 are connected to the computer. The heater 14 is a heating rod, which can be suspended or fixed in the reaction kettle 10 .

Preferably, the second branch 32 is provided with a third branch 33 and its valve at a position away from the pulverizer 22 for passing the mixture that does not require further pulverization. In this embodiment, the pulverizer 22 is a disk-type ultra-fine high-speed shear pulverizer. If the mixture is fine enough, it does not need to pass through the pulverizer 22; at this time, the second branch 32 is provided with a third branch 33 and its valve at a position avoiding the pulverizer 22. The third branch 33 can be seen in Figure 1 The starting point of the path 33 is before the pulverizer 22 , and the end point of the third branch path 33 is after the pulverizer 22 .

Preferably, the second branch 32 is provided with a sampling port 34 so that the reaction status of the mixture in the reactor 10 can be clearly monitored.

Preferably, the blades of the stirring paddle 13 are spiral blades. When the stirring paddle 13 rotates, the spiral blades drive the mixture to rise, and the mixture descends from a position close to the inside of the reaction kettle 10 . The arrows marked in Figure 1 represent the flow state of the mixture. The mixture located in the middle of the reactor 10 is pulled by the spiral blades, while the mixture located at the edge of the reactor 10 naturally descends. This can ensure the uniformity of the material and enzyme during the reaction process, eliminate the temperature gradient within the material and enzyme, thereby improving the reaction speed and reaction efficiency.

Preferably, the thermometer 15 is a thermocouple thermometer.

The method includes the following steps:,

S1. Add water, 150g/L rice starch, and α-amylase with an activity of 15U/g into the reaction kettle 10, adjust the pH value to 6.2 with acid, and add anhydrous calcium chloride at the same time to make the final concentration 2g/g. L.

S2. Start the stirring shaft of the reactor 10 to stir the above mixture. At the same time, start the ultrasonic transducer 16 in the reactor 10 that is submerged in the above mixture. The actual power of the ultrasonic transducer 16 is 100-200w and the vibration frequency is 30kHz. The heater 14 in the reaction kettle 10 is started, the circulation path is started, and the mixture at the bottom of the reaction kettle 10 is first transferred outward, crushed, filtered, and then returned to the reaction kettle 10 .

S3. Heat the mixture to 75°C. When the temperature of the mixture equals 75°C, keep it warm for 15 minutes. Heat the mixture to 90°C and keep it warm until the DE value of liquefaction changes. In order to ensure the stability of the heating process, the heating rate is 2°C/min below 75°C, and the heating rate is 3°C/min above 75°C.

S4. Stop heating when the liquefied DE value reaches 15, and the mixture is cooled to normal temperature while stirring and circulating (when the mixture is cooled, open natural cooling can be used, or a cooling rod can be inserted), and the pH of the mixture is adjusted to 6.0.

S5. Add 0.75U/ml pullulanase, add 2.4U/ml MTSase and MTHase, add α/β-CGTase with a concentration of 1.4U/ml, reheat and incubate at 45°C for 25-30h, adjust The pH of the mixture is 4.5.

S6. Add glucoamylase, raise the temperature to 60°C and keep it incubated for 10-12 hours.

S7. Obtain the product.

The B.subtilis/pHY300PLK-PgsiB-Y, B.subtilis/pHY300PLK-Pxyl-Z and B.subtilis/pHY300PLK-PhpaII-PamyQ'-CGT mentioned above are all from the State Key Laboratory of Food Science and Technology, Jiangnan University .

The fermentation methods of MTSase, MTHase and α/β-CGTase are:

Pick a single colony and culture it in liquid LB (containing neomycin 20ug/ml) for 8-10 hours. Transfer the seed liquid to TB medium (containing neomycin 20ug/ml) according to 5% inoculum volume and shake at 37°C. After culturing in the bed for 2.5 hours, continue culturing on a shaking table at 33°C for about 48 hours. The fermentation broth was centrifuged at 4°C and 8000 r/min for 15 min to collect the bacterial cells, and the cells were concentrated and broken down to obtain crude enzyme solution.

For culture medium:

LB liquid medium: yeast powder is 5.0g, peptone is 10.0g, sodium chloride is 10.0g;

LB solid culture medium: Add 1.5 to 2.0 g of agarose to 100 ml of LB liquid culture medium;

TB medium: peptone is 12.0g, yeast powder is 24.0g, glycerol is 5.0g, KH ₂ PO ₄ is 2.31g, K ₂ HPO ₄ •3H ₂ O is 16.43g.

Based on the above steps, the ultrasonic transducer 16 was eliminated in the comparative example, and no other changes were made, resulting in a product conversion rate of 71.4%.

Based on the above steps, in Example 1, the actual frequency of the ultrasonic transducer 16 is set to 100w, and other parts are not changed, and the conversion rate of the product is 81.6%.

Based on the above steps, in Example 2, the actual frequency of the ultrasonic transducer 16 is set to 150w, and other parts are not changed, and the conversion rate of the product is 82.1%.

Based on the above steps, in Example 2, the actual frequency of the ultrasonic transducer 16 is set to 150w, and other parts are not changed, and the conversion rate of the product is 82.3%.

It can be seen that the ultrasonic-assisted enzymatic synthesis of trehalose has a significant effect on improving the conversion rate of trehalose synthesized from rice starch as the basic raw material.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and illustration. These descriptions are not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teachings. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical applications, thereby enabling others skilled in the art to make and utilize various exemplary embodiments of the invention and various different applications. Choice and change. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims (2)

1. A method for ultrasound-assisted enzymatic synthesis of trehalose, characterized by comprising the following steps: S1. Add water, 150g/L rice starch, and α-amylase with an activity of 15U/g into the reaction kettle, adjust the pH value to 6.2 with acid, and add anhydrous calcium chloride at the same time to make the final concentration 2g/L. ; S2. Start the stirring shaft of the reactor to stir the above mixture. At the same time, start the ultrasonic transducer in the reactor that is submerged in the above mixture. The actual power of the ultrasonic transducer is 100-200w and the vibration frequency is 30kHz. Start the ultrasonic transducer in the reactor. The heater starts the circulation path to transfer the mixture at the bottom of the reactor outward, crush, filter, and then return it to the reactor; S3. Heat the mixture to 75°C. When the temperature of the mixture equals 75°C, keep it warm for a certain period of time. Heat the mixture to 90°C and keep it warm for a certain period of time; S4. When the liquefaction DE value reaches 15, stop heating, stir and circulate the mixture to cool to normal temperature, and adjust the pH of the mixture to 6; S5. Add 0.75U/ml pullulanase, add 2.4U/ml MTSase and MTHase, add α/β-CGTase with a concentration of 1.4U/ml, reheat and incubate at 45°C for 25-30h, adjust The pH of the mixture is 4.5; S6. Add glucoamylase, raise the temperature to 60°C and keep it incubated for 10-12 hours; S7. Obtain the product; The fermentation methods of MTSase, MTHase and α/β-CGTase are: Pick a single colony and culture it in liquid LB containing neomycin 20ug/ml for 8-10 hours. Transfer the seed liquid to TB culture medium containing neomycin 20ug/ml according to 5% inoculation volume and culture it in a 37°C shaker for 2.5 hours. After h, continue to culture in a shaking table at 33°C for 48h; centrifuge the fermentation broth at 4°C, 8000r/min for 15min to collect the cells, concentrate and break the walls to obtain crude enzyme solution; For culture medium: LB liquid medium: yeast powder is 5.0g, peptone is 10.0g, sodium chloride is 10.0g; LB solid culture medium: Add 1.5 to 2.0 g of agarose to 100 ml of LB liquid culture medium; TB medium: peptone is 12.0g, yeast powder is 24.0g, glycerol is 5.0g, KH2PO4 is 2.31g, K2HPO4·3H2O is 16.43g. 2. The method of ultrasonic-assisted enzyme synthesis of trehalose according to claim 1, characterized in that: in step S4, the ventilation port of the reaction kettle is opened to dissipate heat, and the mixture is naturally cooled during stirring and circulation.